Our laboratory is interested in 1) the spinal cord circuitry through which nociceptive information is processed and transmitted rostrally and 2) the brainstem-derived descending control systems which regulate the rostral transmission of nociceptive messages from the spinal cord. By monitoring the expression of the protein product of the c-fos proto-oncogene, we have identified populations of spinal cord neurons that respond differentially to various forms of noxious stimulation. We propose to use double- and triple-label, light microscopic techniques that combine retrograde tracing and fos immunocytochemistry to identify the different brainstem and thalamic first-order targets of noxious stimulus-evoked fos-immunoreactive neurons in spinal cord laminae I, V, VII, VIII and X. We will next test the hypothesis that nociresponsive neurons in these different laminae access different second- and higher-order targets in the brain by combining fos labelling in response to different types of noxious stimuli with the transneuronal retrograde labelling of pseudorabies virus. Of particular interest in these studies are the cortical targets of spinal cord neurons which express the fos protein in response to noxious stimulation. In related studies we will determine whether the analgesia produced by morphine is associated with the selective suppression of fos-immunoreactive projection neurons. To continue our electron microscopic analysis of the circuitry through which opioids exert their analgesic effect, we will focus on noradrenergic synaptology in the spinal cord, by combining pre-embedding immunocytochemistry for tyrosine hydroxylase with post-embedding colloidal gold immunocytochemistry for GABA, glutamate, substance P and CGRP. Finally, we will use in vivo microdialysis to evaluate the factors that regulate the release of GABA from the midbrain periaqueductal gray (PAG) and to test the hypothesis that opioid activation of descending antinociceptive controls involves a lifting of the GABAergic control of the midbrain periaqueductal gray projection neurons. This multidisciplinary approach is directed at providing a comprehensive analysis of the neural substrate of pain and its control.